Substance P-, somatostatin-, vasoactive intestinal peptide- and cholecystokinin-like levels in the spinal cord of polyarthritic rats

Brain Research, 339 (1985) 183-185 Elsevier

183

BRE 20946

Substance P-, somatostatin-, vasoactive intestinal peptide- and cholecystokinin-like levels in the spinal cord of polyarthritic rats S. CHERY-CROZE 1, L. KOCHER 2, C. BERNARD I and J. A. CHAYVIALLE 1 IL~VSERM U45, HOpitalEdouard Herriot, Pavillon Hbis, 69374 Lyon and :Laboratoire de Physiologie, Facult~ de M~dicine Lyon-Sud, LA CNRS 180, 69600 Oullins (France)

(Accepted March 5th, 1985) Key words: substance P - - somatostatin - - vasoactive intestinal peptide (VIP) - - cholecystokinin - - polyarthritic rat - - spinal cord peptide

Substance P-, somatostatin-, vasoactive intestinal polypeptide- and cholecystokinin-like levels were measured in lumbar dorsal and ventral cord of polyarthritic rats and compared with those obtained in vehicle-treated rats taken as controls. Polyarthritis decreased substance P concentration in lumbar ventral cord and increased cholecystokinin level in lumbar dorsal cord, while the other two peptides did not show any change. The results are discussed in relation to immunobistochemical data found in the literature. Polyarthritic rats are considered a useful experimental model because they exhibit symptoms commonly recorded in h u m a n subjects experiencing chronic and intense pain, such as weight loss, irritability4 or hyperventilation 5. Immunocytological, neurochemical and electrophysiological data g a t h e r e d in the last decade suggest that several n e u r o p e p t i d e s could play an i m p o r t a n t role in pain transmission. This contribution has been especially studied in the case of substance P (SP) 9A0'14'20 and there is circumstantial evidence that other peptides involved in pain regulation include somatostatin (ST) 6.11,19, vasoactive intestinal peptide (VIp)2,9,13 and

cholecystokinin (CCK) 7,9,12. The tissue concentration of SP-, ST-, VIP-, and CCK-like immunoreactivity (Li) stored in the spinal cord of polyarthritic rats and of control animals were here m e a s u r e d by r a d i o i m m u n o a s s a y . Polyarthritis was associated with a decrease of SP-Li in the l u m b a r ventral cord and an increase of C C K - L i concentration in the l u m b a r dorsal cord, while ST-Li and VIPLi concentrations were similar in arthritic and control animals. Age- and weight-paired S p r a g u e - D a w l e y rats (n = 14) received a single injection (0.05 ml) at the base of the tail of either heat-killed Mycobacterium

butyricum suspended in paraffin oil (5 mg/ml arthritic, n = 7), or of paraffin oil only (control, n = 7).

During the acute phase of the disease 22 days later, the rats were killed by decapitation between 14.00 and 15,00 h. All animals in the first group presented with important arthritis of hind limbs, while none of the rats in the second group exhibited any sign of arthritis. L u m b a r enlargements were rapidly dissected free and exposed on a cold plate. The ventral and dorsal parts were s e p a r a t e d with a scalpel blade under a magnifying glass. Samples were then quickly frozen on dry ice and stored at - 4 0 °C. F o r extraction, tissue aliquots were weighed out while thawing, then immersed in boiling distilled water for 10 rain and homogenized with a glass tissue grinder (Kontes) in 50 mM potassium p h o s p h a t e , p H 5.5, 100 mM sodium chloride and 5% horse serum, and 50/~g/ml aprotinin. A f t e r centrifugation the supernatant was stored at 4 °C and the precipitate was extracted in 1 N acetic acid for 2 h at 4 °C. The suspension was buffered to p H 6 with 1/10 a m m o n i a , centrifuged at 2000 rpm for 10 min at 4 °C and the supernatant was pooled with the buffer extract. SP-Li was measured with antiserum 81 B (final 1/2,000,000) that recognizes equally the u n d e c a p e p tide and substance p2,~ but poorly the C-terminal

Correspondence: S. Chery-Croze, INSERM U45, H6pital Edouard Herriot. Pavilion Hbis, 69374 Lyon, France.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

184 hexapeptide (inhibitory dose ( I D ) 50 : 0. 1% on a molar basis). STI4, VIP, CCK3.> neurotensin, Met-enkephalin and luteinizing h o r m o n e - r e l e a s i n g h o r m o n e do not react at concentrations >t 100 ng per tube. Synthetic Tyrs-SP (Bachem) was labeled with 1~-sI using lactoperoxydase and purified by reverse-phase high-pressure liquid c h r o m a t o g r a p h y ( # B o n d a p a k Cls, Waters) in isocratic conditions (phosphoric a c i d w a t e r - a c e t o n i t r i l e -- 0.1:74.9:25) as derived from A k a g i et al, ~. With the present extraction p r o c e d u r e , the recovery of 2 5 - 2 0 0 pg SP a d d e d to tissue h o m o genates prior to the acid extraction step was 71 - 110%. ST-Li and VIP-Li were m e a s u r e d as previously described, using respectively antiserum 56 D which recognizes equally ST14 and ST>, and antiserum 76 A that reacts only partially with the C-terminal 18-28 sequence 3, C C K - L i was assayed with antiserum 39A (final 1/200,000) that recognizes equally CCK33 and C C K 8 while the reactivity is 12% for sulfated gastrinw and 5% for non-sulfated gastrinl7 and non-sulfated C C K s. The d e c a p e p t i d e CCK24_33 (gift from E. Wunsch, courtesy of Diamalt Lab, Miinchen) was l a b e l e d with the Bolton and H u n t e r reagent ( m o n o i o d i n a t e d , N E N ) according to F o u r m y et al.S. The mean recovery of 0 . 5 - 2 ng peptide a d d e d to spinal cord h o m o g e n a t e s prior to extraction was 92% for CCK33and 60% for C C K s. Pooled buffer and acetic extracts were tested for the 4 peptides on the same day, at 3 successive dilutions in duplicates. The i m m u n o r e a c t i v e c o m p o n e n t s wcre characterized through gel p e r m e a t i o n of an extract of whole spinal cord on G50 sf Sephadex (1.5 × 100 cm, 10 ml/h at 4 °C). The immunoreactivity was essentially or exclusively d e t e c t e d in the elution volumes of synthetic SP, ST|4, VIP and CCKs, respectively. No gastrin-like immunoreactivity was detected in these elution fractions (sensitivity limit: < 10 pg per fraction). Results were expressed as pg equivalent SP, STI4, VIP and CCK33per mg wet wt. The Student's t-test for unpaired values was used for statistical analysis of the differences between control and arthritic animals. In control rats, the m e a n concentrations of SP-Li and ST-Li (Fig. 1) and of VIP-Li and CCK-Li (Fig. 2) were not significantly different between the ventral and dorsal hemicords. No significant variation of SP-Li and VIP-Li concentrations was record-

CONTROL F ' I ARTHRITIC ~ J - 500

500-

-400

400-

T -300 (/~ t"1 200-

1oo-

O-

l

DORSAL

-2O0"0

-100~

VENTRAL

DORSAL

VENTRAL_

Fig. l. Comparison of SP and ST levels (pg/mg of wet wt.) in vehicle-treated and polyarthritic rats. Bars are S.D. of the mean. *** Significant result P > 0.01.

ed in either l u m b a r hemicord in arthritic rats as compared to control animals. In contrast, arthritis was associated with a 52% decrease of SP-Li in the ventral hemicord ( P > 0.01) and a 40% rise of CCK-Li concentration in dorsal h e m i c o r d (P > 0.005). L e m b e c k et al. 17 observed a significant rise of SP-Li concentrations in the sciatic nerves of arthritic rats, while the increments in T 1 2 - $ 3 sections of dorsal spinal cord, in dorsal root ganglia a n d i n dorsal roots did not reach significance unless the L 4 - L 5 section only, namely where the sciatic nerve enters the cord, was considered18. This may account for the fact that no significant variation of SP-Li in the dorsal cord was here o b t a i n e d , since extracts were p r e p a r e d

CONTROL ARTHRITIC i 60,

-60

5O

-50

~ ,o

-40 -30

2O

(,~ 10

-0 DORSAL

VENTRAL

DORSAL

VENTRAL

Fig. 2. Comparison of VIP and CCK levels (pg/mg of wet weight) in vehicle-treated and polyarthritic rats. Bars are S.D of the mean. * Significant result P > 0.00s

185 from the whole l u m b a r enlargement. A fairly unex-

addition, all CCK-Li positive cell bodies in the dorsal

pected finding was the significant decrease of SP-Li concentration in the ventral l u m b a r cord of arthritic animals. In the ventral hemicord SP is essentially

root ganglia appear to contain SP-Li and vice versa 7. Although both peptides seem to be stored in the same afferent neurones, the present results and those obtained in other u n r e p o r t e d studies suggest that the tissue concentrations of these two peptides may vary

found in fibers of the gray matter, in close vicinity to the cell bodies of m o t o n e u r o n s 10. Konishi and Otsuka 15 have shown that SP is a powerful excitant of motoneurons and induces a potentiation of m o n o s y n a p tic reflexes 16. The observed decrease of SP concen-

independently according to the experimental conditions. The increase in CCK-Li could additionnally be explained by the activation of intrinsic neurones, par-

tration in the ventral hemicord of arthritic rats, could thus represent an indirect mechanism for pain limitation. Indeed, the slightest m o v e m e n t in these animals is likely to evoke an increased activity of nociceptive

ticularly in laminae I I - V 9. Whatever its mecha-

pathways, so that the facilitation of motor reflexes by SP-Li structures in the ventral hemicord would allow for a further increase of pain, detrimental to the animal.

that CCK-like peptides play a significant role in the control of pain mechanisms. From a teleogical point of view, the region-specific variations of SP-Li and CCK-Li here recorded in

nism(s), the rise of CCK-Li tissue concentration observed in the dorsal hemicord of chronically painful rats brings further evidence to support the hypothesis

In the dorsal hemicotd, CCK-Li is mostly concen-

polyarthritic rats may be the basis for a compensato-

trated in laminae I and I1, in fine fibers which have the same distribution pattern as the SP-Li fibers 9. In

ry adaptation to chronic pain, through the limitation of both motor (reflex) activity and pain transmission.

1 Akagi, H., Otsuka, M. and Yanagisawa, M., Identification by high-performance liquid chromatography of immunoreactive SP released from isolated rat spinal cord, Neurosci. Lett., 20 (1980) 259-263. 2 Anand, P., Gibson, S. J., McGregor, G. P., Blank, M. A., Gnatei, M. A., Bacarese-Hamilton, A. J., Polak, J. M. and Bloom, S. R., A VIP containing system concentrated in the lumbo-sacral region of human spinal cord, Nature (Lond.), 305 (1983) 143-145. 3 Chayvialle, J. A., Miyata, M., Rayford, P. L. and Thompson, J. C., Effects of test meal, intragastric nutrients and intraduodenal bile on plasma concentrations of immunoreactive somatostatin and vasoactive intestinal peptide in dogs, Gastroenterology, 79 (1980) 844-852. 4 Colpaert, F. C., Meert, Th., De Witte, Ph. and Schmitt, P., Further evidence validating adjuvant arthritis as an experimental model of chronic pain in the rat, Life Sci., 31 (1982) 67-75. 5 Colpaert, F. C. and Van den Hoogen, R. H. W. N., Ventilatory response to adjuvant arthritis in the rat, Life Sci., 32 (1983) 957-963. 6 Dalsgaard, C. J., H6kfelt, T., Johansson, O. and Elde, R., Somatostatin immunoreactive cell bodies in the dorsal horn and the parasympathetic intermediolateral nucleus of the rat spinal cord, Neurosci. Lett., 27 (1981) 335-339. 7 Dalsgaard, C. J., Vincent, S. R., HOkfelt, T., Lundberg, J. M., Dahlstr6m, A., Schultzberg, M., Dockray, G. J. and Cuello, A. C., Co-existence of cholecystokinin- and substance P-like peptide in neurons of the dorsal root ganglia of the rat, Neurosci. Lett., 33 (1982) 159-165. 8 Fourmy, D., Pradayrol, L., Antoniotti, H., Esteve, J. P. and Ribet, A., Purification of radio-iodinated cholecystokinin peptides by reverse phase HPLC, J. Liq. Chromat., 5 (1982) 757-766. 9 Gibson, S. J., Polak, J. M., Bloom, S. R. and Wall, P. D., The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal, J. comp. Neurol., 201 (1981)

65-79, 10 H6kfelt, T., Kellereth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 11 H6kfelt, T., Elde, R., Johansson, O., Luft, R. and Arimura, A., Immunohistochemical evidence of the presence of somatostatin, a powerful inhibitory peptide, in some primary sensory neurons, Neurosci. Lett., 1 (1975) 231-235. 12 Jeftinija, S., Miletic, V. and Randic, M., Cholecystokinin octapeptide excites dorsal horn neurons both in vivo and in vitro, Brain Research, 213 (1981) 231-235. 13 Jeftinija, S., Murase, K., Nedeljkov, V. and Randic, M., Vasoactive intestinal polypeptide excites mammalian dorsal horn neurons both in vivo and in vitro, Brain Research, 243 (1982) 158-164. 14 Jessel, T. M., Mudge, A. W., Leeman, S. E. and Yash, T. L., Release of substance P and somatostatin, in vivo, from primary afferent terminals in mammalian spinal cord, Soc. Neurosci. Abstr., 5 (1979) 2078. 15 Konishi, S. and Otsuka, M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 397-410. 16 Konishi, S. and Otsuka, M., Excitatory action of hypothalamic SP on spinal motoneurons of newborn rats, Nature (Lond.), 252 (1974) 734-735. 17 Lembeck, F., Donnerer, J. and Colpaert, F. C., Increase of substance P in primary afferent nerves during chronic pain, Neuropeptides, 1 (1981) 175-180. 18 Lembeck, F. and Gamse, R., Substance P in the peripheral sensory processes. In R. Porter and M. O'Connor (Eds.), Substance P in the Nervous System, Pitman, London, 1982, pp. 35-48. 19 Renaud, L. P., Martin, J. B. and Brazeau, P., Depressant action of TRH, LH-RH and somatostatin on activity of central neurones, Nature (Lond.), 255 (1975) 233-235. 20 Sastry, B. R., Substance P effects on spinal nociceptive neurones, Life Sci., 24 (1979) 2169-2178.